Figure 3: A decrease in PI(3,5)P2 by chelating agents suppresses TRPML1 channel activity in the endolysosomal membrane. | Nature Communications

Figure 3: A decrease in PI(3,5)P2 by chelating agents suppresses TRPML1 channel activity in the endolysosomal membrane.

From: PI(3,5)P2 controls membrane trafficking by direct activation of mucolipin Ca2+ release channels in the endolysosome

Figure 3

(a) Post- PI(3,5)P2 quasi-steady state ITRPML1 was inhibited by bath (internal/cytoplasmic) application of poly-lysine (500 μg ml−1) to an enlarged endolysosome from a TRPML1-EGFP-expressing Cos-1 cell. ITRPML1 increased with addition of 100 nM PI(3,5)P2, but gradually reduced to a quasi-steady state level upon washout. (b) Representative traces of ITRPML1 at three time points, as shown in a: upon PI(3,5)P2 application (red), washout (black), and poly-lysine application (magenta). (c) Lack of poly-lysine (500 μg ml−1) effect on whole-endolysosome ITRPML1-Va. (d) Post-PI(3,5)P2 quasi-steady state ITRPML1 was inhibited by bath application of neutralizing anti-PI(3,5)P2 (5 μg ml−1), but not anti-PI(4,5)P2 (5 μg ml−1). (e) Representative traces of ITRPML1 at three time points, as shown in d. (f) Lack of anti-PI(3,5)P2 (5 μg ml−1) effect on whole-endolysosome ITRPML1-Va. (g, h) Large basal pre-PI(3,5)P2ITRPML1 was inhibited by bath application of neutralizing anti-PI(3,5)P2 (5 μg ml−1), but not anti-PI(4,5)P2. (i) ITRPML1 was inhibited by more than 90% by bath (internal/cytoplasmic) application of poly-lysine (500 μg ml−1) or PI(3,5)P2 antibody. Data are presented as the mean±s.e.m.; the n numbers are in parentheses. Statistical comparisons were made using analysis of variance: ***P<0.001.

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